EP0076647A2 - Kulturmedien für Immunitätssystemzellen - Google Patents

Kulturmedien für Immunitätssystemzellen Download PDF

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Publication number
EP0076647A2
EP0076647A2 EP82305187A EP82305187A EP0076647A2 EP 0076647 A2 EP0076647 A2 EP 0076647A2 EP 82305187 A EP82305187 A EP 82305187A EP 82305187 A EP82305187 A EP 82305187A EP 0076647 A2 EP0076647 A2 EP 0076647A2
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Prior art keywords
approximately
acid
cells
group
medium
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EP82305187A
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English (en)
French (fr)
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EP0076647A3 (de
Inventor
Kathelyn Sue Steimer
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Hana Biologics Inc
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Hana Biologics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/40—Nucleotides, nucleosides, bases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components

Definitions

  • the invention relates to culture media, and in particular to media for culturing cells originating from the immune system in serum-free or low serum conditions.
  • hybridomas are usually grown in nutrient medium supplemented with ten to twenty percent serum. Since antibody production by hybridomas is usually in the range of one to one hundred micrograms of immunoglobulin protein per millilitre of culture fluid, the use of serum introduces a vast excess of heterologous protein that must be eventually removed during antibody purification. The development of a serum-free medium for culturing hybridoma cell lines would greatly simplify the task of antibody purification.
  • the present invention provides a culture medium without the drawbacks of the prior art.
  • This medium may be used for the serum-free culturing of a variety of cells originating from the immune system. This includes, in addition to hybridomas, other cell types such as a variety of malignant cells of the immune system and normal lymphocytes.
  • the present invention also provides a culture medium that improves the growth of the above cell types when used with a serum supplement.
  • a culture medium according to the invention has the composition set out in the characterising part of claim 1. This medium is suitable for growing myeloma and hybridoma cell lines, particularly those of murine origin in a serum-free environment.
  • the culture medium according to the invention has the composition set out in the characterizing part of claim 2.
  • the balanced salt solution most preferably comprises the following salts in the following concentrations:
  • the above balanced salt solution is known as Earle's balanced salt solution, as taught by Bryant in Preparation of Saline, in Tissue Culture Association Manual, Vol. 1, No. 4, pp. 185-187 (1975).
  • any balanced salt solution may be used.
  • Hank's balanced salt solution as taught by Hank's & Wallace in Relation of Oxygen and
  • serum is a complex biological fluid containing hundreds of different proteins.
  • the use of serum for hybridoma cell culture introduces a vast excess of protein (approximately 6-30 mg/ml) to the growth medium that must be removed during monoclonal antibody purification.
  • the medium of the present invention contains only three proteins, all purified, and their total maximum possible concentration is only approximately 765 micrograms/ml.
  • Monoclonal antibodies may thus be purified from the defined medium supernatants of the present invention by simple protein fractionization procedures rather than by the more sophisticated techniques such as affinity chromatography that are essential when hybridomas are grown in serum supplemented medium.
  • the medium described herein can be used for growing SP2/0- A g-14 mouse myeloma cells and hybridomas derived from this parent, certain P3-NSI-Ag-4-1 hybridomas, FO mouse myeloma and hybridoma cell lines, and MPC-11 myeloma and hybridoma cell lines. It may also be used for the serum-free culturing of human lymphoblastoid cell lines and human hybridoma cell lines, and as a culture medium for normal lymphocytes. It has been effective for both mitogen and antigen stimulation of proliferation and antibody production by mixed lymphocyte cultures.
  • the immune system may facilitate the purification of effector molecules such as lymphokines or other growth factors produced in vitro by malignant, transformed, and/or normal cells of the immune system either by producing a culture system for obtaining such products in vitro in the absence of added serum or by providing a means for devising an assay system for such effector molecules.
  • effector molecules such as lymphokines or other growth factors produced in vitro by malignant, transformed, and/or normal cells of the immune system either by producing a culture system for obtaining such products in vitro in the absence of added serum or by providing a means for devising an assay system for such effector molecules.
  • the serum-free growth of certain cells may depend upon or be stimulated by such effector molecules.
  • the serum-free culture medium of the present invention can be routinely prepared by the simple addition of each ingredient recited above. Generally, insulin, transferrin, fatty acid linked BSA, antibiotics, glutamine and sodium pyruvate are added immediately before using the medium.
  • the nutrient medium (without the above listed ingredients) is prepared and stored as a liquid at 4-8°C for no longer than 4 months.
  • the basal medium may also be prepared as a dried powder in a manner similar to other conventional tissue culture media, reconstituted with distilled water and filter sterilized.
  • Stocks of insulin in 0.1 N HC1 are prepared at 100-1000 fold higher concentration than in the final recipe and stored at 4°C as a liquid.
  • a 50X stock of transferrin, ethanolamine, and fatty acid linked BSA are prepared and stored frozen.
  • the bovine serum albumin sold under the tradename P entex by Miles Biochemical of Elkhardt, Indiana, is linked in a 1:1 molar ratio with one or more of the following fatty acids: linoleic acid, linolenic acid, palmitoleic acid, oleic acid, arachadonic acid, palmitic acid and stearic acid. More specifically, a solution of 1.5xl- 2 M fatty acid (0.5 ml) in 95% ethanol is added dropwise, while mixing to 10 ml of bovine serum albumin (50 mg/ml) in phosphate buffered saline).
  • the experimental protocol for testing the growth of a particular cell line inthe medium of the present invention was as follows.
  • the cells were detached from their culture flasks by gently pipetting the culture medium over the growth surface or, if the cells were growing in suspension, the cells were gently pipetted to ensure a single cell suspension.
  • the resuspended cells were centrifuged (for example, 800 xg) in a clinical centrifuge, washed once with phosphate buffered saline, and resuspended in phosphate buffered saline. Aliquots of cell suspension were added to the medium of the present invention to yield a final cell density of 2x10 cells/millilitre.
  • Control media tested in parallel were unsupplemented basal medium and basal medium supplemented with 10% fetal bovine serum, basal medium being the present invention without insulin, transferrin, ethanolamine, and fatty acid complexed with BSA. Aliquots of cell suspension were added to 24 well microtiter plates, each well receiving 1 ml of cell suspension. The plates were incubated at approximately 37°C in approximately a 7% C0 2 -93$ air atmosphere. Cultures were monitored daily microscopically and counted after 3 to 5 days. Cell counts were made by using a Coulter electronic particle counter.
  • Cells were serially subcultured in 5 ml of growth medium in 25 cm 2 plastic tissue culture flasks every 3-4 days. The cells were diluted to 2x10 4 cells/ml at each passage. On first passage, the cells were prepared as described above. For later passages, the cells were resuspended and diluted directly into their respective growth media.
  • hybridoma cell lines described in the examples belo were of murine origin and all produced antibody with specificity for keyhole limpet hemocyanin.
  • the culture supernatants from hybridoma cell lines were collected and assayed for levels of IgG antibody with specificity for keyhole limpet hemocyanin by an indirect enzyme linked immunoassay [E LI .SA; Voller, A., D. Bidwell and A. Bartlett (1979).
  • ELISA Enzyme Linked Immunosorbent Assay
  • Polystyrene culture tubes were coated with antigen by adding 0.5 ml of keyhole limpet hemocyanin (20 g/ml) in a 0.1 M carbonate-bicarbonate buffer (pH 9.6). The tubes were incubated at 4°C overnight. At the end of the incubation period, the tubes were washed three times with phosphate buffered saline containing 0.05% Tween 20 and 0.02% sodium azide (PBS-tween), shaken dry, and 0.5 ml of diluted culture supernatant added to each tube. Each culture supernatant was diluted 1/10 and by serial two-fold dilutions thereafter.
  • PBS-tween phosphate buffered saline containing 0.05% Tween 20 and 0.02% sodium azide
  • Dilutions were assayed in duplicate using antigen coated tubes and singly using uncoated tubes.
  • the tubes containing diluted supernatants were incubated at 37°C for two hours. At the end of the incubation period, the tubes were washed three times with PBS-tween, shaken dry and 0.5 ml of alkaline phosphatase conjugated anti-mouse IgG, made in rabbit (Miles-Yeda, Elkhardt, Indiana, cat 561-278) diluted inPBS-tween as recommended by the supplier, was added to each tube.
  • the tubes were incubated for one hour at 37°C, washed three times with pBS-tween and 0.5 ml of lmg/ml p-nitrophenyl phosphate in diethanolamine buffer (10% diethanolamine, 0.02% NaN 3 , 0.01% MgCl 2 .6H 2 O, pH 9.8) was added to each tube. After 30 minutes incubation at 37°C, 0.25 ml of 3N NaOH was added to each tube. The absorbance of each reaction mixture was determined at 405 nm. Dilutions yielding OD 405 readings of 0.4 to 0.7 were used for calculating the reported values. The OD 405 readings of uncoated tubes and negative controls were always less than 0.03 units. The data is expressed as absorbance units per 1 0 4 cells.
  • the medium of the present invention as a culture medium for supporting the growth of and antibody production by murine hybridoma cultures, it was necessary to generate such hybridomas.
  • Balb/c mice were immunized with keyhole limpet hemocyanin, their spleens removed and fused with either SP2/O-Ag-14 mouse myeloma or P3-NS-1-Ag-4-1 mouse myeloma cells.
  • the fusing agent was polyethylene glycol 1500.
  • the methods for cell fusion and hybridoma cell culture are described by Galfre, G., S. How, C. Milstein, G. Butcher, and J. Howard, 1977. Nature 266:550.
  • SP2/0-Ag-14 mouse myeloma cells were cultured in medium of the present invention and basal medium supplemented with 10% fetal bovine serum. Cells were plated at 2x10 4 cells per well in 24 well microtiter dishes and cell counts made daily. The generation time of SP2/0-Ag-14 was approximately 18 hours in the above defined medium and 15.5 hours in basal nutrient medium supplemented with serum SP2/0-Ag-14 cells could be repeatedly subcultured in the medium of the present invention.
  • the generation time of the SP2/O-Ag-14 cells after 12 passages in this serum-free medium was approximately 17 hours, essentially the same as the generation time during the first passage in the medium. It was noted that the SP2/0-Ag-14 cells tended to grow in a suspension as single cells in the serum-free medium rather than loosely attached to the culture flask as they normally do in serum.
  • the importance of insuline, transferrin, ethanolamine, and fatty acid complexed BSA was determined by preparing medium lacking the specific component. It was found that the omission of insulin decreased the final cell density to 65% of that observed in using the above recited formulary of the present invention. Transferrin was absolutely essential for growing SP2/O-Ag-14 cells as without it, there was no growth. The omission of ethanolamine did not significantly affect cell growth while the absence of fatty acid-linked BSA components resulted in cell growth less than 10% of that attained through the use of the entire composition. It was also noted that when a fatty acid free BSA was added instead of fatty acid-linked BSA, the SP2/0-Ag-14 final cell density was less than 5% of that observed in the complete composition.
  • the medium of the present invention developed for culturing SP2/0-Ag-14 mouse myeloma cells also supports the growth of SP2/0-Ag-14 hybridomas.
  • SP2/0-Ag-14 cells and four established SP2/0-Ag-14 hybridoma cell lines producing antibodies to keyhole limpet hemocyanin were cultured in the above described formulary and in basal nutrient medium supplemented with 10% fetal bovine serum. After approximately four days in culture, the SP2/0-Ag-14 cells reached a density in the medium of the present invention that was approximately 85% of that in serum supplemented basal medium.
  • the cell density of the four hybridoma cell lines in the medium of the present invention ranged from 24 to 75% of that attained in serum supplemented basal medium as shown in Table I.
  • A11 hybridomas produce IgG antibodies with specificity for keyhole limpet hemocyanin.
  • the densities reached by the hybridomas were significantly lower in the medium of the present . invention as compared to serum supplemented medium, the increase in cell number over the initial plating value was substantial in each case.
  • the cell density in the medium of the present invention, of the hybridoma cells that grew the slowest of the four cells tested, [S8-2-D12 (F5)7 experienced a thirteen fold increase over their initial plating value.
  • the antibody titers in culture supernatants from all four hybridoma cell lines grown in the serum-free medium were equal to or greater than the antibody titers in serum-supplemented medium as shown in Table II.
  • Each cell line was plated (2x10 4 /ml) in either serum-free or serum-supplemented medium. After four days the supernatants were collected and assayed for antibody with specificity for keyhole limpet hemocyanin by the quantitative ELISA assay. Cell counts were performed at the time that the supernatants were collected.
  • Basal nutrient medium supplemented with 10% fetal bovine serum.
  • T hese hybridomas are producing monoclonal antibodies with specificity for keyhole limpet hemocyanin.
  • SP2/O-Ag-14 myeloma cells grew in suspension in the medium of the present invention. All four SP2/O-Ag-14 hybridomas also grew in suspension in this medium.
  • hybridoma cell lines were serially subcultured in the medium of the present invention for at least seven passages representing approximately 30 to 35 population doublings.
  • One hybridoma cell line Sl-1-D4(E2) was serially subcultured, the growth rate examined periodically, and culture supernatants harvested to be assayed for antibody titers.
  • the generation times in passage 1 in the medium of the present invention and serum-supplemented media were 18 and 17 hours, respectively, while at the twelfth passage, the generation times were 18,5 and 18 hours, respectively. Prolonged passage in the medium of the present invention did not affect antibody production as tabulated in
  • a Sl-1-D4(E2) cells were serially subcultured in either serum-free medium or medium-supplemented with 10% FBS. The supernatants were periodically collected and the levels of antibodies specific to keyhole limpet hemocyanin determined by the quantitative ELISA assay.
  • b M edium of the present invention b M edium of the present invention.
  • c B asal medium supplemented with 10% fetal bovine serum.
  • the levels of keyhole limpet hemocyanin antibodies in the serum-free medium were between 8 and 10 OD 405 units per 10 4 cells, regardless of the passage number.
  • the levels of antibodies in the supernatants collected at various passages in serum-supplemented medium also remained relatively constant although the levels tended to be slightly lower than in the medium of the present invention.
  • the levels of antibodies in the culture supernatants ranged from approximately 5.5 to 8.5 OD 405 units per 10 4 cells.
  • mouse myeloma and hybridoma cells grew well in the media of the present invention.
  • FO a variant of SP2/0-Ag-14
  • the present invention also was an excellent medium for growing MPC-11 mouse myeloma cells.
  • P3-NS-I-Ag-4-lmouse myeloma cells did not grow at all in the medium of the present invention.
  • Two of the three P3-NS-I-Ag-4-1 hybridoma cell lines tested also failed to grow in the serum-free medium.
  • a C ells of each type (2x10 4 /ml) were plated in 24 well microtiter plates in serum-free medium and medium-supplemented with 10% fetal bovine serum.
  • T hese hybridomas are producing antibodies to keyhole
  • the medium of the present invention will also support the growth of human lymphoblastoid cell lines.
  • Four cell lines (BM, AR, 8866 and 8226) all established in culture from patients with multiple myeloma, were cultured in the medium of the present invention.

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EP82305187A 1981-10-02 1982-09-30 Kulturmedien für Immunitätssystemzellen Withdrawn EP0076647A3 (de)

Applications Claiming Priority (2)

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US30787181A 1981-10-02 1981-10-02
US307871 1981-10-02

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EP0076647A2 true EP0076647A2 (de) 1983-04-13
EP0076647A3 EP0076647A3 (de) 1984-02-01

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0112174A2 (de) * 1982-12-16 1984-06-27 Immunex Corporation Serumfreies Zellkulturmittel und Verfahren zur Herstellung desselben
WO1984003710A1 (fr) * 1983-03-24 1984-09-27 Inst Nat Sante Rech Med Milieu de culture de cellules animales sans serum et procedes de culture primaire et d'obtention de lignees cellulaires utilisant ce milieu
FR2604727A1 (fr) * 1986-10-03 1988-04-08 Ceskoslovenska Akademie Ved Milieu synthetique pour la culture d'hybridomes et de cellules de myelome
FR2619576A1 (fr) * 1987-08-20 1989-02-24 Biolog Ind Adjuvants pour la production et la culture d'hybridomes
EP0314496A2 (de) * 1987-10-29 1989-05-03 E.I. Du Pont De Nemours And Company Hybridoma-Herstellung
WO1990012083A1 (en) * 1989-04-12 1990-10-18 Board Of Regents, The University Of Texas System High calcium chemically defined culture medium
WO2001037829A1 (es) * 1999-11-25 2001-05-31 Gomez Moraleda Manuel Antonio Composición que comprende aceites ozonizados y/o otros productos naturales y/o sintéticos ozonizados, y su empleo en composiciones farmacéuticas, cosméticas, dietéticas o de suplementos alimentarios, en los campos humano y veterinario
WO2004101758A3 (en) * 2003-05-09 2005-08-11 Lifeblood Medical Inc Composition for maintaining organ and cell viability
FR2910024A1 (fr) * 2006-12-19 2008-06-20 Agronomique Inst Nat Rech Milieu de culture synthetique chimiquement defini.
US8389267B2 (en) 2009-07-01 2013-03-05 BIOMéRIEUX, INC. Method and culture medium for enhanced detection of Mycobacterium
US8389268B2 (en) 2009-07-01 2013-03-05 BIOMéRIEUX, INC. Method and culture medium for enhanced detection of mycobacterium
US8932851B2 (en) 2009-07-01 2015-01-13 bioMērieux, Inc. Method and culture medium for enhanced detection of Mycobacterium
EP2841561B1 (de) 2012-04-24 2019-01-16 F.Hoffmann-La Roche Ag Zellkulturzusammensetzungen und verfahren zur polypeptidherstellung
CN113512521A (zh) * 2021-05-26 2021-10-19 江苏普瑞康生物医药科技有限公司 无血清培养基的添加剂，无血清培养基及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0756003B1 (de) * 1994-02-18 1999-03-24 Teijin Limited Methode zur kultivierung von tierzellen
JP2018113907A (ja) * 2017-01-18 2018-07-26 株式会社Ｒｅｇｅｎｅ Ｐｈａｒｍ 動物細胞の培養方法

Citations (3)

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Publication number Priority date Publication date Assignee Title
US3128228A (en) * 1960-03-04 1964-04-07 Ustav Ser A Ockovacich Latek Tissue culture medium
US3429867A (en) * 1965-04-30 1969-02-25 Microbiological Ass Inc Serum substantially free from gamma globulin and method of preparing same
US4205126A (en) * 1978-01-01 1980-05-27 Cartaya Oscar A Serum-free cell culture media

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3128228A (en) * 1960-03-04 1964-04-07 Ustav Ser A Ockovacich Latek Tissue culture medium
US3429867A (en) * 1965-04-30 1969-02-25 Microbiological Ass Inc Serum substantially free from gamma globulin and method of preparing same
US4205126A (en) * 1978-01-01 1980-05-27 Cartaya Oscar A Serum-free cell culture media

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0112174A2 (de) * 1982-12-16 1984-06-27 Immunex Corporation Serumfreies Zellkulturmittel und Verfahren zur Herstellung desselben
EP0112174A3 (de) * 1982-12-16 1985-05-15 Immunex Corporation Serumfreies Zellkulturmittel und Verfahren zur Herstellung desselben
WO1984003710A1 (fr) * 1983-03-24 1984-09-27 Inst Nat Sante Rech Med Milieu de culture de cellules animales sans serum et procedes de culture primaire et d'obtention de lignees cellulaires utilisant ce milieu
US4786599A (en) * 1983-03-24 1988-11-22 Institut National De La Sante Et De La Recherche Medicale Serum-free animal cell culture medium and methods for the primary culture and production of cell lines using this medium
FR2604727A1 (fr) * 1986-10-03 1988-04-08 Ceskoslovenska Akademie Ved Milieu synthetique pour la culture d'hybridomes et de cellules de myelome
FR2619576A1 (fr) * 1987-08-20 1989-02-24 Biolog Ind Adjuvants pour la production et la culture d'hybridomes
EP0305278A1 (de) * 1987-08-20 1989-03-01 Biologie Et Industrie Adjuvanzien für die Herstellung und Kultur von Hybridomen
EP0314496A2 (de) * 1987-10-29 1989-05-03 E.I. Du Pont De Nemours And Company Hybridoma-Herstellung
JPH01153085A (ja) * 1987-10-29 1989-06-15 E I Du Pont De Nemours & Co ハイブリドーマ増殖用培地
EP0314496A3 (de) * 1987-10-29 1989-08-30 E.I. Du Pont De Nemours And Company Hybridoma-Herstellung
WO1990012083A1 (en) * 1989-04-12 1990-10-18 Board Of Regents, The University Of Texas System High calcium chemically defined culture medium
US5126261A (en) * 1989-04-12 1992-06-30 Board Of Regents, The University Of Texas System High calcium chemically defined culture medium
WO2001037829A1 (es) * 1999-11-25 2001-05-31 Gomez Moraleda Manuel Antonio Composición que comprende aceites ozonizados y/o otros productos naturales y/o sintéticos ozonizados, y su empleo en composiciones farmacéuticas, cosméticas, dietéticas o de suplementos alimentarios, en los campos humano y veterinario
ES2162586A1 (es) * 1999-11-25 2001-12-16 Moraleda Manuel Gomez Composicion que comprende aceites ozonizados y/o otros productos naturales y/o sinteticos ozonizados, y su empleo en composiciones farmaceuticas, cosmeticas, dieteticas o de suplementos alimentarios, en los campos humano y veterinario
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